Life?s wear and tear can leave joints damaged. All too often, the result is the pain and disability of osteoarthritis (OA). Because pain is the most debilitating symptom of OA, it remains the primary target for therapeutic interventions that typically progress from non-steroidal anti-inflammatory drugs (NSAIDs) to weak opioids, and then to stronger opioids. If pain can?t be controlled, total joint replacement surgery remains the only viable long- term treatment option. However, the associated risks and costs will necessarily keep surgery an option of last resort. Thus, there is a critical need for the development of safe and effective methods for the treatment of OA- associated pain. Recently, our team successfully developed an in vitro multi-component joint on a chip (microJoint), in which engineered osteochondral complexes, synovium and adipose tissues were integrated. OA-like pathology has also been successfully modeled in the microJoint. In this new grant application, we propose to introduce sensory innervation into the microJoint. The result will be a more ?complete? joint that enables the dynamic interplay between the peripheral nervous system and joint tissues. We hypothesize that a distinct combination of factors released from different cellular/tissue compartments within the joint mediate pain, hypersensitivity, and hyper- innervation of OA. Furthermore, we hypothesize that opioids not only increase the rate of joint degeneration but potentiate the release of pain producing mediators. In aim 1, a neuron-containing microfluidic ally will be developed to innervate the current microJoint (Neu-microJoint). This new bioreactor will be 3D printed and allow for the non-invasive assessment of neural activity via multi electrode arrays embedded in the tissue chamber, and high-speed optical recording. Human sensory neurons or induced pluripotent stem cell (iPSC)-derived sensory neuron progenitors, will be cultured in the new bioreactor chamber. In aim 2, our previsouly establised OA-model will be created in the Neu-microJoint system. We will assess the activation and/or sensitization of nociceptive afferents with electrophysiology, as well as neurite outgrowth. In aim 3, we will mechanically insult the Neu- microJoint and assess the emergence of ?pain? in response to prolonged mechanical stress of the joint with the goal of creating a more natural OA-model. In aim 4, we will assess the impact of drugs used clinically for the management of OA on our OA models in the Neu-microJoint as a means of validating this platform for the assessment of therapeutic efficacy and toxicity of novel compounds. We will then use ?omic? approaches to identify new biomarkers and therapeutic targets. Results from this unbiased screen may not only reveal an injury specific pain signature, but suggest medications approved for use in people that could be re-purposed for the treatment of OA pain. In aim 5, we will assess the impact of opioids on neural activity in the presence and absence of joint injury triggered with different stimuli, as well as the integrity of all joint elements. Our Neu-microJoint will enable identification of mechanisms responsible for pain associated with joint injury and therefore novel therapeutic targets, screening of therapeutic interventions, and the implementation of personalized therapeutic strategies.